Modular micro-PCR system for the onsite rapid diagnosis of COVID-19

The COVID-19 pandemic necessitates rapid and accurate virus detection. Real-time reverse transcription-polymerase chain reaction (RT-PCR) remains the gold standard, but the need for accessible, rapid, and cost-effective point-of-care (POC) tests is crucial for widespread testing and pandemic control. Existing commercial solutions, such as Cepheid® Xpert® Xpress SARS-CoV-2, Roche’s cobas® SARS-CoV-2, and GenMark ePlex® SARS-CoV-2, offer high sensitivity but are expensive and bulky. Other POC tests, like COVIDNudge and Abbott ID NOW COVID-19, offer portability but may have limitations in accuracy and sensitivity. Microfluidics offers advantages for POC testing due to its use of small sample volumes and reduced reagent needs. While some microfluidic PCR systems exist, they often lack modularity or efficient temperature control, hindering their deployment in low-resource settings. This research addresses these challenges by presenting Epidax®, a modular microfluidic platform designed for rapid and cost-effective COVID-19 diagnosis.

The literature review highlights the existing limitations of commercial and POC COVID-19 diagnostic tests. High-sensitivity, automated systems like Cepheid Xpert Xpress and Roche cobas are expensive and lack portability, limiting their use in resource-constrained environments. Other portable options such as Abbott ID Now, while fast, have demonstrated accuracy and sensitivity issues. The review also discusses the challenges in developing microfluidic PCR systems, such as achieving efficient temperature control and modularity for various testing applications. Existing microfluidic PCR platforms, while promising, often lack the scalability and ease of use needed for widespread implementation. This gap in the market motivates the development of Epidax.

Epidax® is a modular platform comprising a temperature module, a microfluidic chip, a detection module, and analysis software. The chip, easily mounted and self-aligned using magnets, is disposable while the temperature module is reusable, reducing costs. The on-chip temperature module utilizes a Peltier element, spacer plate, and support plate, with an aluminum adhesive film enhancing thermal conductivity and fluorescent signal. The chip's design includes microchannels with serpentine regions for mixing and an eye-shaped chamber for the reaction mix, optimizing temperature control and preventing sample breakup. Various channel configurations are possible, enabling flexibility in testing volume and number of samples. The detection module contains a CMOS camera, LED light source, and filter cube for fluorescence signal capture. In-house software processes images for real-time or endpoint detection, providing amplification plots and Ct values for real-time PCR or comparing pre- and post-amplification images for endpoint PCR. Epidax was tested using endpoint RT-PCR, real-time RT-PCR, and RT-LAMP on clinical RNA extracts and nasopharyngeal samples, comparing results to a commercial platform.

Epidax® demonstrated high sensitivity, detecting as low as 1 RNA copy per µL for both real-time and endpoint RT-PCR, while using only half the reagents compared to commercial systems. In comparative studies using 81 clinical RNA extracts, Epidax® showed results comparable to a commercial platform for both endpoint and real-time RT-PCR assays. The platform's efficiency was further highlighted by its ability to perform direct RT-PCR on 44 nasopharyngeal swab samples without prior RNA extraction, reducing the sample-to-answer time to approximately one hour. Further validation using an RT-LAMP assay on 54 clinical RNA extracts confirmed the platform's performance. The modular design of Epidax® allows easy reconfiguration for various testing scenarios and sample types, highlighting its versatility and potential for wide-scale deployment.

The findings demonstrate that Epidax® offers a sensitive, cost-effective, and accurate alternative to existing COVID-19 diagnostic platforms, particularly suitable for low-resource settings. The platform's modularity and ease of use simplify testing procedures, reducing the time and expertise needed for sample processing and analysis. The ability to perform direct RT-PCR on nasopharyngeal samples without RNA extraction significantly streamlines the workflow and accelerates results. The comparable performance to commercial systems, coupled with the reduced reagent consumption and lower cost, makes Epidax® a compelling solution for addressing the challenges of large-scale COVID-19 testing.

Epidax® provides a significant advancement in COVID-19 diagnostics by offering a sensitive, cost-effective, and modular microfluidic platform. Its performance is comparable to existing commercial systems while significantly reducing reagent usage and complexity, making it well-suited for various settings including those with limited resources. Future research could explore further miniaturization, integration of additional functionalities (e.g., sample preparation), and application to other infectious diseases.

While Epidax® shows promising results, limitations include the need for further large-scale clinical validation across diverse populations. The current study is limited to a specific set of clinical samples and may not represent the full range of viral loads encountered in real-world scenarios. Long-term stability of the reagents and the platform's performance under various environmental conditions need further investigation.